ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response

Abstract

Nijmegen breakage syndrome (NBS) is characterized by extreme radiation sensitivity, chromosomal instability and cancer. The phenotypes are similar to those of ataxia telangiectasia mutated (ATM) disease, where there is a deficiency in a protein kinase that is activated by DNA damage, indicating that the Nbs and Atm proteins may participate in common pathways. Here we report that Nbs is specifically phosphorylated in response to gamma-radiation, ultraviolet light and exposure to hydroxyurea. Phosphorylation of Nbs mediated by gamma-radiation, but not that induced by hydroxyurea or ultraviolet light, was markedly reduced in ATM cells. In vivo, Nbs was phosphorylated on many serine residues, of which S343, S397 and S615 were phosphorylated by Atm in vitro. At least two of these sites were underphosphorylated in ATM cells. Inactivation of these serines by mutation partially abrogated Atm-dependent phosphorylation. Reconstituting NBS cells with a mutant form of Nbs that cannot be phosphorylated at selected, ATM-dependent serine residues led to a specific reduction in clonogenic survival after gamma-radiation. Thus, phosphorylation of Nbs by Atm is critical for certain responses of human cells to DNA damage.

Cleavage of the BRCT tandem domains of nibrin by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation

“…In particular, after IR treatment, ATM phosphorylates NBN at Ser278 and Ser343 (21)(22)(23)(24). These two Ser residues are crucial for the proper activation of the intra-S phase checkpoint in response to the DNA damage (46).…”

“…Although ATM responds to the presence of DSBs, ATR appears to be activated by single-stranded DNA, arising at stalled replication forks or generated during processing of bulky lesions (53,54). ATR phosphorylates many of the same damage response proteins as ATM, including NBN (21)(22)(23)(24), which localizes to stalled replication forks (55).…”

“…Notably, the BRCT domains are the major mediators of phosphorylation-dependent protein-protein interactions in cell cycle checkpoint and DNA repair mechanisms (14)(15)(16)(17)(18)(19)(20). The central region of NBN contains a consensus sequence encompassing the Ser343 residue that, together with the Ser278 residue located within the BRCT2 domain, is phosphorylated by ATM kinase in response to IR (21)(22)(23)(24). Lastly, the C-terminal region of NBN contains the MRE11-binding domain and the ATM-binding motif (12,25).…”

Cell cycle regulation of the human Six1 homeoprotein is mediated by APCCdh1

“…Purified proteins (25-50 mg) (still attached to the resin) were incubated in interphase or mitotic extracts from Xenopus laevis oocytes (a gift from Todd Stukenberg) for 30 min at room temperature. After incubation, the proteins were electrophoresed on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, appropriate bands were cut from the gel, trypsinized, and subjected to ion trap mass spectrometry (Wu et al, 2000) by William S Lane at the Harvard Microchemistry and Proteomics Analysis Facility. Protein coverage of the Six1 protein incubated in the interphase extract was 53%, whereas coverage of the Six1 protein incubated in the mitotic extract was 36%.…”

Chromatin modification and NBS1: their relationship in DNA double-strand break repair

“…Most of the substrates that are phosphorylated by ATM have consensus sequences with SQ/TQ motifs, and these motifs are also found in the central region of NBS1. Specifically, serine residues at positions 278 and 343 are phosphorylated by ATM in response to radiation both in vitro and in vivo (Lim et al, 2000;Wu, X. et al, 2000), and are highly conserved in vertebrates (Tauchi et al, 2002b). Substituting these phosphorylation sites with alanine residues resulted in the abrogation of the ATM-dependent intra-S checkpoint in response to DSB damage in mammalian cells, which was also observed in the cells of NBS patients (Lim et al, 2000;Wu, X. et al, 2000).…”